U.S. patent number 5,874,668 [Application Number 08/547,521] was granted by the patent office on 1999-02-23 for atomic force microscope for biological specimens.
This patent grant is currently assigned to Arch Development Corporation. Invention is credited to Morton F. Arnsdorf, Shaohua Xu.
United States Patent |
5,874,668 |
Xu , et al. |
February 23, 1999 |
**Please see images for:
( Certificate of Correction ) ** |
Atomic force microscope for biological specimens
Abstract
An atomic force microscope for quantitative imaging and
identification, at the molecular or submolecular level,
biomolecules or subunits of biomolecules in a physiologic
environment, through use of a cantilever tip incorporating a
biomolecular identifier.
Inventors: |
Xu; Shaohua (Chicago, IL),
Arnsdorf; Morton F. (Chicago, IL) |
Assignee: |
Arch Development Corporation
(Chicago, IL)
|
Family
ID: |
24184977 |
Appl.
No.: |
08/547,521 |
Filed: |
October 24, 1995 |
Current U.S.
Class: |
73/105; 977/853;
850/40; 850/59 |
Current CPC
Class: |
G01Q
60/42 (20130101); B82Y 35/00 (20130101); Y10S
977/853 (20130101) |
Current International
Class: |
G12B
21/00 (20060101); G12B 21/08 (20060101); G12B
21/02 (20060101); G01B 005/28 () |
Field of
Search: |
;73/105 ;250/306 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Hoh et al., "Atomic Force Microscopy For High-Resolution in Cell
Biology", Miscellenean. .
Nakagawa et al., "Discriminating Molecular Length of Chemically
Adsorbed Molecules Using an Atomic Force Microscope Having a Tip
Covered With Sensor Molecules (An Atomic Force Microscope Having
Chemical Sensing Function).", Jpn. J. Appl. Phys. vol. 32, Part 2,
No. 213, 15 Feb. 1993, pp. L294-L296. .
Specht et al., "Simulteneous Measurement of Tunneling Current and
Force As a Function of Position Through a Lipid Film on a Solid
Substrate", Surface Science Letters, vol. 257, 1991, pp.
L653-L658..
|
Primary Examiner: Williams; Hezron E.
Assistant Examiner: Larkin; Daniel S.
Attorney, Agent or Firm: Rechtin; Michael D. Foley &
Lardner
Claims
What is claimed in:
1. An atomic force microscope for probing a biological specimen,
comprising:
a cantilever arm having a cantilever tip attached thereto, said
cantilever tip including means for identifying biomolecules on the
surface of said biological specimen with said means comprising an
amphipathic molecule coupled to said cantilever tip for interaction
with a biological site of interest;
means for providing a laser beam for reflecting off said cantilever
tip;
means for scanning said cantilever tip relative to said biological
specimen; and
means for detecting said reflected laser beam.
2. The atomic force microscope as defined in claim 1 wherein said
amphipathic molecule is selected from the group consisting of small
lipid molecules, detergents, and large macromolecules.
3. The atomic force microscope as defined in claim 2 wherein said
amphipathic molecules are selected from the group consisting of
charged molecules and zwitterions.
4. The atomic force microscope as defined in claim 1 wherein said
cantilever tip comprises at least one leg portion and at least one
of a hole and a cutout in said leg portion.
5. The cantilever tip as defined in claim 4 wherein said leg
portion includes a plurality of holes.
6. The cantilever tip as defined in claim 5 wherein said cantilever
tip comprises at least two of said leg portions.
7. The cantilever tip as defined in claim 6 wherein said leg
portions have different patterns of said holes.
8. The atomic force microscope as defined in claim 1 wherein said
tip comprises a ceramic material.
9. The atomic force microscope as defined in claim 1 wherein said
amphipathic molecule has a pKa near a pH to be measured.
10. An atomic force microscope for probing a biological specimen,
comprising:
a cantilever arm having a cantilever tip attached thereto, said
cantilever tip including means for identifying biomolecules on the
surface of said biological specimen wherein said means for
identifying biomolecules comprises at least one of receptor
molecules coated onto said cantilever tip, a zwitterion liquid
coating, amphipathic liquids having protein donors, amphipathic
liquids having protein acceptors, and hydrophobic molecules;
means for providing a laser beam for reflecting off said cantilever
tip;
means for scanning said cantilever tip relative to said biological
specimen; and
means for detecting said reflected laser beam.
11. An atomic force microscope for probing a biological specimen,
comprising:
a cantilever arm having a cantilever tip attached thereto wherein
said cantilever tip is coated with photoactivated charges, said
cantilever tip including means for identifying biomolecules on the
surface of said biological specimen;
means for providing a laser beam for reflecting off said cantilever
tip;
means for scanning said cantilever tip relative to said biological
specimen; and
means for detecting said reflected laser beam.
Description
The present invention is concerned generally with atomic force
microscope systems. More particularly, the invention is concerned
with atomic force microscope subsystems, including an electrostatic
microscope, an electroprobe microscope and an ultrasoft microscope
probe and methods of use of these subsystems.
Biologic surfaces are the interfaces to the outside world. The cell
surface with its specialized biomolecules including receptors,
channels and pumps is where most regulatory hormones, drugs and
other signals are directed, thereby making the biologic surface of
particular interest to physiologists and pharmacologists. In order
to shed light on these problems, it would be important to: (1)
image and identify biomolecules at a molecular level in a
physiologic environment; (2) visualize dynamically the
structure-function relationships of biomolecules during
physiologic, molecular biological, and pharmacologic perturbations;
(3) physically manipulate biomolecules; and (4)
electrophysiologically perturb, while imaging and/or measuring
forces, voltage-sensitive tissues, and biomolecules. An instrument
that could provide these capabilities would be invaluable in the
study of the physiology and pharmacology of excitable tissues and
biological molecules including those of the heart, vasculature,
brain and nervous system, gastrointestinal system including the
pancreas, and many other systems in living sciences.
By applying to an electrophysiologic instrument the principles of
scanning probe microscopy (SPM), particularly atomic force
microscopy (AFM) and scanning tunneling microscopy (STM),
substantial insight in biological environments can be gained.
Achievement of such discoveries has been frustrating because of the
relatively poor resolution and/or the destructive nature of
available modalities for imaging surface topology. For example, the
electron microscope, although capable of high resolution, is
limited because of the necessity of viewing the specimen in a
nonphysiologic vacuum. The imaging of biological membranes and
biomolecules, then, has been elusive. To date, emphasis has been on
static structure, but physiologists and pharmacologists are more
interested in the dynamic structure of a protein associated with
ligand binding and dissociation under physiologic conditions.
Efforts are being made to assess structure and function using
crystallized proteins, but it is uncertain whether crystallized
structure is the same as in the native membrane and whether normal
physiologic function is preserved. Every technique has its
strengths and limitations, and the strengths of AFM are that it can
obtain structural information with high resolution at the protein
surface where the ligand binding and dissociation occur. Further,
the sample can be analyzed under controlled physiologic
conditions.
In terms of pragmatic matters, sudden cardiac arthythmic death is a
major public health problem accounting for perhaps 300,000 or more
fatalities each year. Studies suggest that abnormal cardiac
excitability is required to produce the proarrhythmic state
responsible for malignant cardiac arrhythmias. Cardiac excitability
is determined by the activities of biomolecules that are in, or
span, the membrane including receptors, ion channels, gap junctions
that mediate cell-cell communication, and pumps.
FM has been used primarily in industry to assess the uniformity of
materials, the molecular structure of bulk organic materials and
the characteristics of microchips. Compared to what is required for
biological imaging, however, this is very low power resolution. The
application to biology has been limited, and AFM in fact is not
used very much in the biological research community. AFM has been
used with varying success by some researchers to image statically:
amino acid crystals (which are the building blocks of biological
structures), proteins, genetic material (including DNA and RNA),
organic monolayers and bilayers, whole cells, planar membranes and
membrane bound proteins and lipid bilayers such as those that make
up the membrane of the cell, as well as artificial membranes that
are often used to reconstitute proteins of importance. Dynamic AFM
imaging has been limited, but used enough to demonstrate the
potential of AFM, particularly the imaging of fibrin
polymerization, which is the process that occurs when blood clots,
of the interaction of important chemical pathways in the body such
as the phosphorylase-phosphorylase kinase system, and, as
mentioned, the formation of antigen-antibody complexes.
There are also a number of limitations with AFM imaging, and two
particularly important limitations include: (1) calibration of the
tip and distortion due to the interaction between the tip and
specimen; and (2) the unambiguous identification of imaged
structures and recognition of binding sites. The AFM image results
from an interaction between the tip and the specimen. A major
criticism of AFM is the lack of calibration, which can detect
distortion and estimate errors in measurement, that results from
varying geometry and characteristics of the scanning tip.
One of the remaining substantial problems in using AFM for
biological imaging in the living sciences is the need to
unambiguously identify imaged structures and biomolecules and to
locate with certainty the important biologically active subunits of
such cells and biomolecules such as receptors and binding sites.
Biomolecules are soft and easy to compress, often floating in a sea
of lipids so that the cantilever tip may force the biomolecule down
into the bilayer; in addition, the biomolecules are small. The
distortion of images, the creation of artifactual images, and
inaccuracies in measurement due to the shape of the scanning tip
are relatively unimportant in the material sciences, but have posed
major problems in imaging biomolecules. Biomolecules and the
membranes in which they often exist are also fragile structures.
The commonly used Si.sub.3 N.sub.4 tip exerts a force of
approximately 0.1 to 10 nN on the specimen; and an important
concern has been whether this force causes structural damage.
Therefore, a particular major limitation of the use of AFM in
biological studies has been the stiffness of the imaging probe
which, in turn has limited the resolution of the image. The probe
also is known to compress biological specimens and likely produces
mechanical injury to some of the biological specimens under
study.
It is therefore an object of the invention to provide an improved
atomic force microscope and method of use.
It is also an object of the invention to provide a novel passive
probe having molecular level resolution of biological
environments.
It is another object of the invention to provide a novel method and
apparatus for high resolution detection of structural changes
through imaging and/or force measurements when a cell is voltage-
or current-clamped internally and as the cells are exposed to
changing external fields or environments which alter transmembrane
potentials or chemical and/or biochemical conditions in and out of
the cell.
It is an additional object of the invention to provide an improved
apparatus and method for mapping biological fields using an atomic
force microscope with fuctionalized cantilever probes acting as
sensors for biological environments.
It is still another object of the invention to provide a novel
method and apparatus for mapping and identifying receptors and
biomolecules of physiologic and pharmacologic importance with high
spatial resolution.
It is yet a further object of the invention to provide an improved
method and apparatus for high resolution imaging of biological
media in solutions which improve resolution and allow study of
dynamic physiologic and pathophysiologic conditions after
structural or chemical perturbation.
It is still also an object of the invention to provide a novel
method and apparatus for identifying physiologically and
pharmacologically important biomolecules and their subunits on a
cell surface using highly sensitive force measurements.
It is also an object of the invention to coat the cantilever tip
with membrane reconstituted receptors, ion channel proteins, and
other proteins for drug screening.
It is yet an additional object of the invention to provide an
improved method and apparatus for controllably moving ligands,
proteins, and other biological markers at the nanometer level to
measure interactive forces.
It is also a further object of the invention to provide a novel
atomic force microscope cantilever tip having a high degree of
flexibility and selectable structure to control the cantilever tip
degrees of freedom to respond.
It is another object of the invention to provide a novel group of
chemicals for coating a cantilever tip of an atomic force
microscope to control reactivity, charge state and density.
It is a further object of the invention to provide an improved
method and apparatus for imaging biological receptor structures,
while simultaneously recording the single- or multichannel
conductance.
It is still an additional object of the invention to provide a
novel method and apparatus for examining effects of cellular
transmembrane potential on binding forces of materials on the
cantilever tip of an AFM and on interaction forces between ligands,
hormones, agonists and antagonists, and receptors.
It is also another object of the invention to construct the two
legs of a cantilever tip with different spring constants to enhance
the sensitivity of frictional force or lateral force imaging.
It is also another object of the invention to provide an improved
method and bioprobe apparatus for delivering drugs to a specified
molecule while monitoring interactions and resulting effects of
delivery.
It is yet a further object of the invention to provide a novel
method and apparatus for selectively coating an atomic force
microscope cantilever tip with liquid coatings to form salt
bridges, with amphipathic lipids to monitor surface hydrogen bonds,
with hydrophobic materials to monitor surface hydrophobicity, and
with photoactivated charges and electrical conductivity coatings
for selected measurements.
It is still another object of the invention to provide an improved
method for manufacturing atomic force microscope cantilever tips to
any geometric shape or combinations of shapes.
These and other objects and advantages of the invention will become
apparent from the following description including the drawings
described hereinbelow.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A illustrates an atomic force microscope constructed in
accordance with one form of the invention; and FIG. 1B illustrates
scanning microscope and AFM operating modes using a scanning probe
tip;
FIG. 2A illustrates a cantilever tip, laser and detector part of an
atomic force microscope and FIG. 2B illustrates a schematic of a
fluid cell which allows imaging in a physiologic environment;
FIG. 3A shows an untreated AFM scanning probe tip; FIG. 3B shows a
treated tip allowing binding to other molecules; FIG. 3C shows a
tip fuctionalized by attaching a particular material to the tip;
FIG. 3D shows a biospecific molecule attached to the tip and near a
specific binding site; FIG. 3E shows a biospecific molecule on a
tip joined to a specific site; and FIG. 3F shows a biospecific
molecule attached directly to the tip;
FIG. 4A shows an AFM modified to perform electroprobe
functionalities in accordance with the invention; FIG. 4B shows a
current flow record for a scan by the modified AFM; FIG. 4C shows
an AFM image illustrating cell channels for the point "t" on FIG.
4B; FIG. 4D shows two patch clamp electrodes of the AFM positioned
on the surface of two cardiac myocytes; FIG. 4E shows recorded data
of the voltage dependent decay of junctional currents from the
electrodes of FIG. 4D and FIG. 4F shows selected current tracings
with single channel activity of the gap junction for different
junctional voltages;
FIG. 5 illustrates hydrophobic force between an untreated Si.sub.3
N.sub.4 tip and a paraffin surface with (1) being measured in water
in the presence of 5M NaCl (5M NaCl, 2 mM CaCl.sub.2, 1 mM EDTA),
(2) in water in the presence of ethanol and (3) in water in the
absence of the 5M NaCl of (1);
FIGS. 6A and B illustrate various adhesion forces between a
negatively charged pG tip and a positively charged surface with
FIG. 6A imaging DEAE-Sephadex beads and (1) being the DEAE-Sephadex
placed on a mica surface and imaging with electrostatic force
microscopy using the pG tip and adhesion force marked F'.sub.adh,
(2) being the same tip as (1) imaging mica under water or (3) same
tip being coated with pG used to image DEAE-Sephadex beads and FIG.
6B imaging APTES-glass in water with (1) being the adhesion force
between the pG tip and the APTES-glass, F.sub.adh ; (2) being
APTES-glass imaged after the lipids had been washed away from both
the tip and a fluid cell and (3) being APTES-glass imaged with the
same tip before being coated with pG, F.sub.a'h ;
FIGS. 7A and B show adhesion and attractive forces between a
positively charged DOSPA tip and mica with FIG. 7A illustrating
adhesion force for mica imaged under water with (1) being a DOSPA
tip and (2) being a bare tip and FIG. 7B illustrating attractive
force with an extending force curve shown and (1) being a DOSPA tip
on mica in water; and (2) being a naked tip on mica in water;
FIG. 8 shows stability of the DOSPA tip; the adhesion force was
recorded as a criteria for the stability of the lipid tip with the
DOSPA tip found to be stable imaging mica over time (top trace
result was compared to the naked tip on mica (bottom trace) and the
paraffin surface (middle trace); the adhesion forces recorded here
were measured in water; and for the DOSPA tip, the final lipid
concentration was 1.5 .mu.g/ml;
FIGS. 9A and B illustrate probing of the surface charges of
lysozyme with a pG tip; a pG tip was used to image lysozymes on
mica under phosphate buffer (10 mM sodium phosphate, 2 mM
CaCl.sub.2, 1 mM EDTA, pH 7.0); FIG. 9A shows an image of the
lysozyme and FIG. 9B shows the adhesion forces measured between a
naked tip (the dashed line) and the lysozymes and between a pG tip
and the lysozymes (the open circles); the same naked tip was used
for the preparation of the pG tip; and FIG. 9C shows a schematic
representation of formation of a lipid tip;
FIG. 10 shows a model of cellular gap junction structure;
FIGS. 11A-D illustrate atomic force dissection responses for
increasing forces of 0.8 nN, 3.1 nN, 10.1 nN and a repeat scan at
10.1 nN and FIG. 11E shows the line cut for each image;
FIGS. 12A and B show models of nicotine ACHR receptor with the five
subunits and active binding site for ACh; FIG. 12C shows an AFM
image of the AChR showing the five subunits; FIG. 12D shows two of
the M2 segments of the ion channel with three negatively charged
rings drawing positively charged ions through the channel;
FIG. 13A shows a V-shaped AFM tip; and FIGS. 13B-D show the
V-shaped tip with various orifices in the tip generated by laser
beam removal of material;
FIG. 14A shows a force versus distance curve generated by an AFM (a
"force curve"); FIG. 14B shows the force curve and the bend of the
cantilever at forces equal, greater and less than zero for a
positively charged tip and negatively charged mica in water; FIG.
14C shows forces generated by withdrawal of the sample from the
cantilever and disengagement for a positively charged tip (DOSPA)
and a positively charged surface (mica plus DEAE-Sephadex to render
the scanned surface positive); FIG. 14D shows only forces occurring
on withdrawing the sample surface from the tip which was a
negatively charged (pG tip) and negatively charged (mica) and a
positively charged (DEAE-Sephadex treated); FIG. 14E shows only
forces recorded on withdrawing the sample from the tip (untreated)
imaging mica in the presence and absence of 5M NaCl; FIG. 14F shows
only forces recorded on withdrawing sample from the tip with a
nonpolar paraffin sample, imaging performed in ethanol, water and
5M NaCl.
FIG. 15 is a schematic illustration of the tip geometry
deterioration and SFM image calibration, in accordance with the
present invention and for use in conjunction therewith; and
FIG. 16A is a three-dimensional plot of a tip structure from a
representative analysis of gold particles; and FIG. 16B is a
comparison plot.
SUMMARY OF THE INVENTION
The present invention includes an atomic force microscope for
probing a biological specimen. Microscope components include (1) a
cantilever arm having a tip attached thereto, with the tip
including means for identifying biomolecules on the surface of the
biological specimen, (2) means for providing a laser beam for
reflecting off the cantilever tip, (3) means for scanning the tip
relative to the biological specimen, and (4) means for detecting
the reflective laser beam. Useful in conjunction with this
invention are means for generating an electronic signal
characteristic of the tip position, means operating on the signal,
and means generating data as described more fully below.
Alternatively and with reference to the following, the microscope
of this invention can include various biomolecular identifiers for
use in conjunction with the cantilever tip. In preferred
embodiments, the means for identifying biomolecules and/or the
biomolecular identifiers of this invention include a chemical
entity for selective interaction with a biological site of interest
on this specimen.
The chemical entity can include but is not limited to an antibody,
a ligand, and a protein. Alternatively, the chemical entity
includes an amphipathic molecule coupled to the cantilever tip.
Such amphipathic molecules can be selected from but are not limited
to the small lipid molecules, detergents, and large macromolecules.
Alternatively, such amphipathic molecules can be various charged
molecules and/or zwitterions.
Likewise, the microscope of this invention can include means for
identifying biomolecules and/or biomolecular identifiers of the
type disclosed herein, which have, without limitation one or more
of the following: receptor molecules coated onto the cantilever
tip, a zwitterion liquid coating, amphipathic liquids with protein
donors, amphipathic liquids with protein acceptors, and hydrophobic
molecules.
Without limiting the scope of this invention, other embodiments
include atomic force microscopes with cantilever tips coated with
photoactivated charges, conductive materials, or semiconductive
materials.
In part, the present invention can also be described as a
cantilever tip suitable for use in a probe for sensing biological
surface structure. The tip includes at least one leg portion and
either one or both of a whole or a cutout in the leg portion. In
various preferred embodiments of the invention, the tip has a leg
portion which includes a plurality of holes. In various highly
preferred embodiments, the tip can include at least two leg
portions and, alternatively, leg portions with different hole
patterns. Notwithstanding any one particular tip configuration, the
probe suitable for use therewith can include but is not limited to
an attractive force imaging probe, an adhesion force imaging probe,
and a mechanical repulsive force imaging probe.
In part, the present invention also includes an atomic force
microscope for electrical probing of a biological specimen. Such a
microscope includes (1) a cantilever arm having a tip attached
thereto, (2) means for coupling electrodes to a biological specimen
and means coupled thereto for applying selected electrical fields
and sensing electrical outputs responsive to, (3) means for
providing a laser beam for reflecting off the cantilever tip, (4)
means for scanning the tip relative to the biological specimen, and
(5) means for detecting the reflected laser beam. In various
preferred embodiments, the tip includes a pharmaceutical coating
and, alternatively, a pharmaceutical coating combined with a
biological site on the biological specimen.
An improved atomic force microscope (AFM) constructed in accordance
with the invention is indicated generally at 10 in FIGS. 1A and 1B.
The AFM 10 passively senses interatomic force through a cantilever
12 (see FIG. 2A) or arm that has a very sharp imaging tip.
Conceptually, this can be thought of as an atomically sharp
phonograph needle (tip 14) that tracks across the surface of a
material 16. The tip 14 detects the contours and bumps in the
grooves of the "phonograph," and the motion of the needle (or the
tip 14) and the phonograph arm (the cantilever 12) is translated
into an electrical signal that is converted to sound.
As shown in FIG. 1B in the case of the AFM 10, the scanning probe
tip 14 of the AFM 10 is only a few atoms in size, so the forces
that surround these atoms interact with the forces in a material or
specimen 16 that surround the atom or conglomeration of atoms in
molecules of the specimen 16 under study (primarily repulsive
forces in the AFM 10 as shown on the right, but it can be the
forces of the electron cloud that surrounds the atoms as with
another form of this passive force microscope, the scanning
tunneling microscope 18 (STM) on the left). As the tip 14 scans the
surface of the sample (FIG. 1; S in FIG. 2A), the interatomic
forces deflect the tip 14 and hence the cantilever 12. The
cantilever 12 (see FIG. 2A) to which the scanning probe tip is
attached is the force sensor. The cantilever spring has a spring
constant that is lower than the effective spring between two atoms.
The deflection is measured by a laser optical deflection detection
system, and FIG. 2A shows a laser source 20, a laser beam 22
passing through an optical system 24 that is reflected off the
cantilever 12 onto two photodetectors 26 and 28. The reflected
laser beam 22 falls asymmetrically at the two photodetectors 26 and
28. A piezo tube on which the specimen resides moves the sample in
the x, y, and z dimensions and is also used in a feedback loop to
keep the deflection and (hence the tracking force) constant while
the tip 14 scans the specimen 16. The specimen 16 is scanned in the
X- and Y-planes with this cantilever 12, the deflection in the
Z-plane is measured, the signals converted by electronics 13 to a
signal for display, and the resulting three-dimensional contours
are displayed on a computer screen 30. FIG. 1 shows x,y raster 31
and the cantilever tip 14 moving towards and away from the surface
as well as a three dimensional picture on the computer screen 30.
FIG. 2B is a schematic of a fluid cell 32 that allows imaging in a
physiologic environment which, as will be discussed, is one of the
unique advantages of the AFM 10. The fluid cell 32 is sealed by use
of an "O" ring seal, and both the cantilever tip 14 and the
specimen 16 (in this case, labeled "gap junctions") are immersed in
a fluid that can be changed through inlet and outlet ports.
Bioprobe
In a preferred form of the AFM 10, a specifically functionalized
cantilever tip (a bioprobe tip 52 as shown in FIG. 3) allows the
identification of physiologically and pharmacologically important
biomolecules and their constituent subunits. This preferred form of
the AFM 10 can be used on pure preparations, on preparations
reconstituted in artificial membranes and on the cell surface of
living cells. In essence, the bioprobe tip 52 can be
biospecifically functionalized and will interact specifically with
a molecule or site on the molecule while force is being measured
and an image obtained.
An antibody, ligand or other protein or chemical that is capable of
interacting specifically to a biomolecule or site on a biomolecular
of interest is attached to the bioprobe tip 52). For example
amphipathic molecules, such as small lipid molecules, detergents or
large macromolecules (including polymers or proteins), can be used
to coat the tip 14. These molecules can be charged or zwitterions.
The tips 14 can be coated in the manner set forth in Example I and
II. By mixing different compositions of ampipathic molecules, one
can control the charge density on the tip 14 using molecules with
different PKAs of the acidic or basic group. A negatively charged
tip 14 can be converted readily to a positively charged tip 14 by
changing the solution pH (which changes the polarity and charge).
Coating materials can readily be removed by washing the tip 14 and
the test chamber with organic solvents, such as chloroform or
ethanol. This enables reuse of the tips 14 without
recalibration.
When the bioprobe tip 52 comes in contact with the molecule or
subunit of the molecule to which the tip 52 has been specifically
functionalized, a strong force would be needed to separate them
(rupture force) due to the interaction, and the increased force of
the interaction can be measured using the AFM 10. The force of
interaction between the bioprobe tip 52 and the sample 16 is
measured while scanning with the AFM 10 to obtain both force and
morphological information.
We have found that "force dissection" can be used to suggest the
type of adhesion forces present. We not only can measure the force
of attraction, but we can also measure the force required to
separate the bioprobe tip 52 from the specimen 16 which estimates
the force of the binding between molecular pairs.
Referring to FIGS. 3A-F, the bioprobe tip 52 begins as an untreated
scanning probe tip (A) which is then treated to alter the charge
(B), a chemical compound, e.g., a lipid, is attached to the treated
surface (C), a biospecific molecule 34 is attached to chemical 32
or, alternatively, can be attached directly to the treated scanning
probe tip 52, and the biospecific molecule 34 acts as a biospecific
probe, binding, for example, a receptor 38.
In FIG. 3A, the scanning bioprobe tip 52 of the cantilever 12 is
the component that is functionalized to become biospecific. We have
been using as a base tip a standard commercial microfabricated
V-shaped Si.sub.3 N.sub.4 cantilever with an integrated tip (of
Si.sub.3 N.sub.4). The choice of this tip 14 was its availability
and its compatibility with standard atomic force microscopes. A
number of other materials can be used so long as their surface
charge can be changed, and some other materials may be suitable
with minimal or no surface modification.
In FIG. 3B, the surface of the tip 14 is treated so that it can
bind other molecules 32. We have used several strategies to alter
the surface of the cantilever tip 14 by modifying the charge
(positive or negative charge as shown) or the stickiness of the
surface, and other approaches.
In FIG. 3C, the surface of the tip 14 that has been treated can be
fuctionalized either directly (see F below where the biospecific
molecule 34 is attached to the tip 14) or through an intermediate
structure that is attached to the tip.
In FIG. 3D, the biospecific molecule 34, for example a protein that
can interact only with a specific receptor, is attached to chemical
32 (shown) or directly to the scanning probe tip (not shown). The
biospecific molecule 34 is shown approaching the receptor 38 which,
in turn, is in the membrane of a cell.
In FIG. 3E, the biospecific molecule 34 recognizes the specific
receptor 38 (or other specific site such as an antigen, antibody,
etc.) in this case in cavities on the probe molecule that contain
anionic groups that react with the cationic groups of the receptor
38, a possible H binding site, or other mechanisms of binding.
There will be a strong force of attraction due to the binding of
the ligand probe (the biospecific molecule 34) and the receptor 38
and the increased force of the interaction can be measured using
the AFM 10.
In FIG. 3F, the surface of the tip 14 that has been treated often
can be functionalized either directly by attaching the biospecific
molecule 34 directly to the tip 14 itself as shown in this figure.
The fuctionalized tip 14 would react with the receptor 38 or other
specific site on the receptor 38 or other protein as in FIG.
3E.
This phenomenon of interaction can include, for example, use of a
monolayer or multilayer of lipids coated onto the tip 14 (see FIG.
9C) for studies of membrane lipid structure and chemical or
physical properties or electrophysical properties of membranes.
Biomolecules which bind periplasmically or anchor into the
molecules can be coated onto the tip 14 for study of chemical and
biochemical properties. Receptors 38 can be coated onto the tip 14
to screen its interaction with different drug molecules coupled to
the specimen 16, or between a membrane and a cell or virus, or DNA
protein.
In another aspect of this invention the tip 14 (and even the
cantilever 12) can be coated photoactivated charges with activation
accomplished using the laser beam 22 (see FIG. 2A). A conductive
line can even be disposed on the cantilever 12 by activating part
of the photoactivated charges, washing off the unactivated part by
conventional photolithography techniques, including deposition of a
secondary coating as needed to complete a narrow conductive line on
the cantilever 12.
For the reasons discussed above, the bioprobe tip 52, coupled with
the AFM 10, offers the highest resolution available for physiologic
biological imaging, and can be done in physiologic solutions (with
minimal or no cellular damage).
Electroprobe Microscope (EPM)
In another preferred form of the AFM 10, electrophysiologic
perturbations can be applied to biological materials and studied.
FIG. 4A illustrates an electroprobe microscope (EPM) 100
constructed in accordance with the invention. The EPM 100 includes
most of the components of the AFM 10 of FIG. 1 which has been
described above. The components of the EPM 100 in FIG. 4A include a
piezoelectric XY scanning arm 40 to which is attached the
cantilever 42 that can be lowered and positioned utilizing a
commercially available controller. A laser optical system 44 is
used in which a laser beam 46 reflects off the cantilever 42 to
photodetector 48. A deflection of the cantilever 42 causes the
reflected laser beam 49 to fall asymmetrically onto the
photodetector 48. The difference signal from the photodiodes are
operated on by electronics 50; and the output is used, most
commonly, as a feedback loop to keep the deflection and hence the
tracking force constant while the tip 14 scans the specimen 16. The
piezoelectric arm 40 rasters the specimen 16 in the X- and
Y-planes, and the Z-plane is superimposed resulting in a
three-dimensional scanner on the CRT or other display device (not
shown). The EPM 100 can therefore function to generate a current
flow record for a scan (FIG. 4B) with an AFM image in FIG. 4C
generated for cell channels for the point "t" in FIG. 4B.
FIG. 4D shows two patch clamp electrodes 52 that have been
positioned on the surface of two cardiac myocytes 54. Suction was
applied through a port (not shown) to form a gigaseal and rupture
the cell membrane thereby allowing voltage control. The two
headstages allow collection of the electronic signal and current
can be fed back through these electrodes 52 to accomplish current-
or voltage-clamping. Both cells (the myocytes 54) can be
individually voltage-clamped. The microelectrode arrangement
enables control and test solutions to be injected into the
micropipette and thereby into the cell manually,
electrophoretically or with a stepper motor. FIG. 4E shows
recordings of the voltage-dependent decay of junctional currents
(i.sub.j) from a study in adult rat atrial cells. FIG. 4F shows
selected tracings of currents with single-channel activity of the
gap junction for different junctional voltages. Single myocytes and
multicellular cardiac tissues as well as neurological preparations
can be studied with the EPM 100. Complicated multistep
voltage-clamp protocols can be programmed into the computer. With
reconstituted membranes and excised patches, a potential difference
can be created across the membrane using this arrangement. The
effect of transmembrane potential on binding force of materials
coated on the tip 14 can be monitored. This will help understand
how the membrane potential affects adhesion force of water soluble
molecules onto cell surfaces. Further, the EPM 100 enables imaging
receptor structure and recording single and multiple channel
conductance simultaneously. Voltage regulated in channels can be
activated while imaging their structural changes in both open and
closed states on the cell surface in physiological solutions.
In the EPM 100 of FIG. 4A, we include capabilities of current- and
voltage-clamping of single cells, patches, and artificial
membranes. The voltage-clamping capabilities include patch clamping
as well as single and multiple microelectrode techniques. The
positioning of the electrode(s) 52 necessary for voltage
perturbation, control and measurement is also shown in FIG. 4A.
Access to the specimen 16 is required for both the probe and the
electrodes 52 (or field electrodes in the case of reconstituted
membranes). As seen in FIG. 4A, the design utilizes an inverted
microscope 58 that allows the preparation to be visualized
optically and by video camera 60 from below through a transparent
tissue bath 62. This cellular electrophysiologic arrangement also
allows the use of specialized light sources when voltage-sensitive
and other dyes are used. The temperature of the tissue bath 62 can
be controlled either by radiant heat since continuous superfusion
will be disruptive to the imaging. The solutions can be changed by
utilizing input and output ports. The design of the tissue bath 62
shown (F) is for whole cell (or two-cell) voltage-clamping. A small
modification utilizing a support can be substituted for the tissue
bath 62, and we have an insert that orients the reconstituted
membrane as desired. The solution within the micropipette can be
modified using standard techniques. We can even introduce
antibodies and drugs into the tips 14 as part of an experimental
protocol in which voltage is applied and electrical response
characterized.
The probe being used as a sensor is important to the EPM 100. Study
of the functionalized cantilevers 42 was started by analyzing
force-distance curves generated by imaging a variety of substrates
and samples with a commercially available microfabricated
"V"-shaped 100-200 micron Si.sub.3 N.sub.4 cantilever with an
integrated tip (of Si.sub.3 N.sub.4) (Digital Instruments, Santa
Barbara, Calif.). A variety of attractive, repulsive forces and
adhesive forces act on the cantilever tip 14. In normal contact
mode imaging, interatomic repulsive forces (due to overlapping
electron clouds) predominate and are responsible for images. Long
range attractive forces include coulombic and van der Waals forces.
Adhesion forces include short range coulombic, hydrophobic, van der
Waals, capillary and hydrogen bonding forces. These cantilever
probes that have been functionalized to recognize specific proteins
such as a channel or subunits of the protein will be most useful in
guiding the imaging cantilever 42. The AFM circuitry allows
nanometer positioning control.
Software useful in conjunction with the invention and, in
particular, for operating the EPM 100 will be available to a
programmer of reasonable skill and made aware of the invention,
through straight-forward modification of existing commercial
software capable of image analysis. Without limiting the invention
in any manner, any such program can be guided by one or more of the
following considerations: Integration of controller and
electrophysiologic data; timing of electrophysiologic events with
imaging so as to allow the proper structure-activity correlations;
accurate positioning and restriction on the number of scans so as
to increase the temporal resolution to 5 msec or less which is
required for most voltage-sensitive biomolecules (one dimensional
scanning will require image direction, similar to the use of
two-dimensional echocardiography that will allow the selection of a
single line for analysis; a series of single dimensional images
could be reintegrated into an image); and superimposition of images
with force measurements.
Irrespective of the particular software, programming, signal
generation and/or data acquisition systems utilized, it is
contemplated that the tip will be calibrated to account for and
reconcile tip geometry and its interaction with the specimen
structure. A protocol of the type required for correction of the
tip effect, from SFM images, will include two steps: (1)
calculation of the true tip structure as illustrated schematically
in FIG. 15 (steps A-C); and (2) deduction of the specimen structure
from the SFM image which is a tip-sample convolution product, as
illustrated in FIG. 15 (steps D-F). The scanning of the tip over
the spherical colloidal gold particles will generate a picture of
(B), which will be a convolution product of both the tip and the
gold particle. Elimination of the volume of the gold particle will
yield the structure of the portion of the tip interacting with the
gold particle. The gold particle is assumed to be an ideal
spherical. The highest point of the image of the gold particle is
assigned to be the diameter of the gold particle and to be the
tallest point of the particle. The resulting tip is in an upside
down orientation which is expected from the path of the tip or the
S-line. Assuming (E) is the image of a specimen obtained using the
same tip calibrated with gold particle, the true size and shape of
the specimen can then be calculated as shown in (F).
For gold particles with a size of 40 nm or less, the tip structure
dominates the image. For purposes of illustrating this aspect of
the type or kind of programming which could be used, FIG. 16A is a
three-dimensional plot of an image of a 20 nm gold particle and the
tip structure from the analysis of the image with software
representative of the type which can be used with this invention
and, particularly, for tip reconstruction. The irregular pattern of
the images of the spherical gold particles observed are a result of
the unpredictable shape of the tip. For purposes of comparison,
FIG. 16B is a three-dimensional plot of an image of 20 nm gold
particle imaged with a bad tip and the tip geometry reconstructed
from the image of the same 20 nm gold particles.
AFM technology permits high resolution imaging of living cells in a
physiologic environment. The modification that will allow the
electrophysiologic control of voltage-sensitive biomolecules will
allow the study of structure-activity and structure-function of
biomolecules that are central to the control of the brain, nervous
system, heart, the pancreas, the gut and other structures in health
and disease. The EPM 100 will delineate how pharmaceutical agents
act. The use of fuctionalized biospecific cantilevers allows the
precise identification of voltage-sensitive structures.
The advantages of the EPM 100 are therefore that it can obtain
quantitative structural information with high resolution at the
membrane protein surface where the ligand binding and dissociation
occur, the sample 16 can be analyzed under physiologic conditions,
and, the fuctionalized biospecific cantilever tips 14 will allow
the measurement of interaction forces and, thereby, the unambiguous
identification of molecules and subunits of molecules as well as
the binding sites of specific ligand and drug molecules. The EPM
100 can be used to study many important voltage-sensitive
biomolecules.
The precise positional control of the EPM 100 is an important
advantage so that the probe can be brought to an individual
molecule. Moreover, the force of the cantilever 12 against the
specimen 16 can be used to "dissect" molecules or structures, as,
for example, in our studies on gap junctions in which "force
dissection" revealed normally inaccessible surfaces, specifically,
the docking areas of the gap junctions that allow cell to cell
communication (see FIGS. 10-12).
Electrostatic Force Microscope (EFM)
Untreated Si.sub.3 N.sub.4 tips ("naked tips"), were used as a
reference. These reference tips 14 were then treated so as to
render them positively or negatively charged and also to image
substrates that were positively charged, negatively charged or
nonpolar. Moreover, imaging could be performed in air or, using a
fluid cell, in a variety of solutions. Hypotheses based on current
theory were tested by analyzing force-distance curves which
provides insight into the interaction between tip and specimen.
FIG. 5 illustrates a force (y-axis) vs. distance (x-axis) curve
generated by the EFM, which hereafter for simplicity will be called
a "force curve."
The standard Si.sub.3 N.sub.4 tip had a hydrophobic surface as
evidenced by a force curve determined from the image of a paraffin
surface. A high adhesion force (9.6 nN) was measured when a naked
Si.sub.3 N.sub.4 tip imaged the paraffin surface (dielectric
constant of 2.2) in water (trace 1, FIG. 5). The force became
negligible when the imaging was performed in ethanol (0.2 nN, trace
2 in FIG. 5) and only slightly decreased when performed in 5M NaCl
solution (9.8 nN, trace 3 in FIG. 5). In comparison, when a mica
surface was imaged with an untreated or "naked" Si.sub.3 N.sub.4
tip, a five- to ten-fold reduction of the adhesion force was
observed when imaged in 5M NaCl instead of water. The coulombic and
the van der Waals forces seemed to play a significant role on the
adhesion force between the naked tip and the paraffin surface since
the addition of 5M NaCl, which would change the dielectric constant
of the media, had little effect on the adhesion force. Both
coulombic and van der Waals force are inversely related to the
dielectric constant of the solution.
One of the tips 14 coated with pG acted as a negatively charged
sensor. The adhesion forces between the negatively charged pG tip
14 (net negative charge per pG molecule is 1) and the cationic
DEAE-Sephadex beads was 12.7 nN (trace 1, FIG. 6A), much higher
than the 0.1 nN observed for a naked tip on the beads or the pG tip
on mica (traces 2 and 3, FIG. 6A). Ca.sup.++ (up to 4 mM) increased
the adhesion force by one- to five-fold for both the naked tip 14
and the pG tip 14 imaging mica.
Imaging APTES-treated glass showed a higher adhesion force with the
negatively-charged pG tip 14 (16.2 nN, trace 1, FIG. 6B) as
compared to the naked tip 14 (2.5 nN, trace 2) or the pG tip 14
after lipids had been washed away (2.1 nN, trace 3). The
measurements with a naked or organic solvent-cleaned tip 14 were
essentially the same.
A cationic DOSPA tip 14 (five net positive charges per lipid
molecule) yielded high attractive and adhesion forces on negatively
charged mica (5.4 and 44.5 nN) for the attractive and adhesion
forces, respectively, traces 1 in FIGS. 7A and 7B, as compared to a
bare tip (0 and 0.5 nN for the attractive and adhesion forces,
traces 2 in FIGS. 7A and B). Both the sizes of the attractive and
the adhesion forces were lipid concentration dependent. A five- to
eight-fold reduction of the adhesion force was observed for
different lipid tips 14 when imaged in 5M NaCl solution (5M NaCl, 2
mM CaCl.sub.2, 1 mM EDTA) as compared to imaging in water.
Three different tips 14 were analyzed for their geometry before
being used for the force study (Table 1), and the adhesion forces
for the naked tips 14 on paraffin and mica and the DOSPA tips 14 on
mica varies. The first and second tip 14 with similar R.sub.T seem
to have similar adhesion forces on paraffin (without tip coating)
and mica (with DOSPA coating), whereas the third tip 14 with larger
R.sub.T showed higher adhesion force on paraffin and mica.
TABLE 1 ______________________________________ Tip parameters and
forces for four tips under various circumstances Tip Parameter 1 2
3 ______________________________________ R.sub.T, nm 40.5 .+-. 1.0
44.5 .+-. 1.0 75.2 .+-. 1.0 .alpha., .degree. 52.6 .+-. 0.5 60.2
.+-. 0.5 83.0 .+-. 1.0 F.sub.p, nN (n = 30) 9.7 .+-. 0.6 11.6 .+-.
1.0 22.3 .+-. 0.5 F.sub.m, nN (n = 30) 16.6 .+-. 0.6 15.3 .+-. 0.3
31.8 .+-. 0.5 F.sub.m ', nN(n = 30) ND ND 0.6 .+-. 0.1
______________________________________ R.sub.T and .alpha.,
curvature radius and semivertical angle of the tip, respectively,
determined as described. F.sub.p and F.sub.m ', adhesion forces for
naked tips imaging paraffin and mica, respectively. F.sub.m,
adhesion forces between DOSPA tips and mica surface. ND, not
determined.
The DOSPA tip 14 was stable as shown in FIG. 8 in which the
adhesion forces of a naked tip 14 on mica (bottom line) and
paraffin (middle line) and the DOSPA tip 14 on mica (top line)
remained constant over one minute of sampling (31.7.+-.0.4). Tips
14 coated with DOSPA were tested up to two hours and the adhesion
forces remained constant (31.6 nN.+-.0.6, n=150).
Lysozymes adsorbed on mica were imaged in phosphate buffer using a
pG tip (FIG. 9A). A spectrum of adhesion forces between 4 and 9 nN
was recorded for the interaction between the pG tip 14 and the
lysozymes which was much higher than the 0.5 nN obtained for the
interaction between the naked tip 14 and lysozymes under the same
conditions (FIG. 9B).
Surface charges on a nanometer scale in aqueous solutions can be
readily analyzed by the method and apparatus of the invention. In
evaluating the effect of surface charges, phospholipids were
preferentially used to coat the tip 14 because: (1) different
lipids with different charges could be applied to a tip
sequentially or simultaneously; (2) they hold a promise as a base
for the attachment of small chemicals such as drug molecules and
small ligands and, more importantly, they will allow a substrate
for reconstituting membrane proteins such as receptors and ion
channels onto the tip 14 for pharmacological studies; and (3) the
lipids could be removed from the tip 14, allowing different studies
with the same cantilever tip 14 which eliminated the errors
generated using tips with different tip geometry and cantilever
spring constants.
The following nonlimiting discussion elaborates on the advantages
and possible scientific bases of the observations. An electrical
double layer can exist on the surface of a Si.sub.3 N.sub.4 with
ionizable groups resulting from partial oxidation including Si--OH
and Si.sub.2 --NH. Depending on the pH and the electrolyte
concentration, the surface charge of silicon nitride can be either
positive, zwitterionic (zero net charge) or negative. At neutral
pH, the net charges on the silicon nitride surface are near zero.
The percentage of the charged to uncharged area of the tip surface
rather than the nearly neutral charge status of the tip 14 is more
important to the orientation of the phospholid molecules. Under our
experimental conditions (pH, 6.0 and 7.0) the charged area may only
represent a small percentage of the total surface since the
adhesion force between an untreated tip 14 and a paraffin surface
(nonpolar, hydrophobic) was ten times larger than the force
measured on mica. This suggests the force of interaction between
the tip 14 and the hydrophobic tails (nonpolar, hydrophobic) of the
lipid molecules is much higher than the interaction between the tip
and the head groups (polar, charged). The lipid molecules probably
oriented themselves so that the hydrophobic tails were in contact
with the tip 14, and the head groups were probably facing the
aqueous solution (see FIG. 9C). This seems likely after the silicon
nitride tip makes contact with a preformed lipid monolayer at the
water-air interface. The structure can be more complicated or can
become more complicated (trilayers, multilayers) as the result of
interactions during imaging or the heterogeneity of binding to the
tip 14. The bottom line, however, is that our measurements were
reproducible over time and did not seem to be much affected by any
potential or real change in the structure of the lipid molecules on
the tip during imaging.
The attractive force between the DOSPA tip 14 and mica was probably
due to coulombic and van der Waals forces. The adhesion force was
due to coulombic, van der Waals forces, and more importantly, the
force needed to break the salt bridges and hydrogen bonds formed
between the lipid molecules on the tip and the specimen. The
surface of mica is negatively charged in aqueous solution with a
charge density of 0.009 C/m.sup.-2 or 0.056 charges/nm.sup.2 (27)
due to the dissociation of the alkaline ions. A lipid molecule
occupies an area of approximately 6 nm.sup.2. Roughly one salt
bridge could be formed for every three lipid molecules between the
DOSPA on the tip 14 and mica.
Long-range electrostatic double layer repulsive force was observed
for naked tip 14 imaging both mica and paraffin in aqueous
solution. Strong attractive force observed for DOSPA tip 14 on mica
was probably the sum of the long range electrostatic attractive
force between the two oppositely charged surfaces and the short
range van der Waals force.
Dications are known to be able to stabilize the monolayer
structure. The effect of calcium on the adhesion force could result
from a structural change of the lipid monolayer on the tip 14,
since a change in the surface potential on both the specimen 16 and
the monolayer surface of the tip 14 and a change in the dielectric
constant of the media due to the addition of Ca.sup.++ would only
decrease the adhesion force.
A legitimate concern of using lipids rather than chemicals bound
covalently to the tip 14 is whether the lipid molecules could be
stripped off the tip 14 during scanning. Although possible, it did
not seem to affect the use of the lipid tip 14 to monitor surface
charges. We tested DOSPA-tips 14 on mica surface and the force
remained almost unchanged for a fixed position over two hours of
continuous monitoring of the adhesion force. Potentially, it might
be of benefit if the lipids were pulled off the coated tip surface.
The tips 14 could be designed to allow a ligand to be delivered to
the protein receptor (the biospecific molecule 38) to initiate the
biological function of the receptor molecule.
The adhesion force varied significantly when the pG tip 14 was used
to interact with different particles of DEAE-Sephadex. This
contrasts to the rather uniform force measurements when the DOSPA
tip 14 interacted with different areas of mica. Such variation
could result from nonuniform charge distribution on the sephadex
beads or perhaps from changing areas of tip-specimen contact due to
the uneven surface of the sephadex beads.
The variation of the adhesion forces in the experiment with
lysozymes might reflect different areas of the molecule being
probed. The force between the pG tip 14 and the protein receptor 38
might not necessarily result from interaction with a single
molecule. However, it would be reasonable to believe that the
molecule forming salt bridges with the pG tip 14 contributed more
to the adhesion force than the other lysozyme molecules.
Whenever an SFM image is obtained in the presence of membrane
lipids (such as cells, membrane proteins and artificial membranes)
or other surfactant in aqueous solution, a charged form of the tip
14 can be formed from the interaction of the tip 14 with the
monolayer of lipids or surfactant on the water-air interface. The
danger is that we may be actually imaging the specimen 16 with a
charged or surface active form of the tip 14 instead of the
relatively inactive Si.sub.3 N.sub.4 tip 14. As long as the lipids
or surfactant existed in the aqueous solution, those amphipathic
molecules could make the tip surface more active by either forming
a monolayer or double layer depending upon the surface chemistry of
the original tip 14. Energetically, it is a favorable process to
make the tip surface more active since it would mean less
structurally ordered water molecules around the hydrophobic tip 14
and an increase of entropy.
The idea of using amphipathic molecules to form charged tips can be
generalized to the preparation of any other functionized tips. This
can be a potentially powerful approach to studying biomolecules of
physiologic and pharmacologic importance. As long as the materials
used to modify the tip 14 have a hydrophobic tail and a polar head
group, one could chemically modify the head group and attach other
small chemical molecules to it. It should be practical to link drug
molecules or ligand molecules as well as other agonist or
antagonists to the lipids and prepare drug and ligand forms of the
tip 14. One can, for example, use an amphipathic molecule which has
a pKa around the pH to be measured to form an amphipathic tip and
to access the surface pH in a nanometer scale.
Ultrasoft Probe
In use of the AFM 10, one can further enhance performance and
develop new degrees of freedom by utilizing a modified ultrasoft
probe tip. In FIG. 13A is illustrated a conventional Si.sub.3
N.sub.4 tip 150. The ultrasoft probe tip is a very flexible
cantilever tip used as the probe for imaging with the atomic
(scanning) force microscope (AFM). The process that creates the
ultrasoft probe tip 152 can use laser technology, or other means
for introducing holes 154 or other cutouts 156 from the tip 152.
(See FIGS. 13B-D.). These holes or spaces of various shapes in the
tip 152 of the AFM 10 increase the flexibility of the tip material
while retaining the necessary strength for imaging. The ultrasoft
probe tip 152 will decrease the imaging probe's spring constant
thereby decreasing the loading or tracking force. The decreased
tracking force, in turn, will increase the spatial resolution of
the AFM 10 and decrease the compression of and the potential
mechanical damage to a biological specimen. The process can be used
with Si.sub.3 N.sub.4 as in our prototype or with other
materials.
Spatial resolution depends importantly on a number of
characteristics of the cantilever and its tip 152. The cantilevers
12 with lower spring constants certainly will reduce the loading
force on the specimen 16 and will reduce the compression of and
potential mechanical injury to biological specimens.
A strategy for improving resolution is to decrease the tracking
force of the cantilever 12 with a force less than or equal to
10.sup.-10 N which is thought to be in the range required for
nonperturbed biological imaging. A reduced loading or tracking
force will decrease the compression and potential mechanical damage
of the biological or other fragile specimen 16.
The AFM imaging probe has been made of a variety of materials. The
ultrasoft probe tip 152 is preferably made of Si.sub.3 N.sub.4, the
material most commonly used in commercially available cantilevers,
but the tip 152 can be constructed from a large number of suitable
materials such as refractory materials, metals, ceramics,
semiconductors, superconductors, plastics and other materials. FIG.
13A shows the detail of the narrow short-legged "V" shaped or
triangular portion of the cantilever tip that is the "spring" of
the imaging probe. The legs are approximately 200 .mu.m in length
and 15 .mu.m in width. The "spring" need not have this "V" shape,
and the process described applies to all shapes and most materials.
Further, the legs can have different spring constants, allowing
friction force imaging or lateral force imaging taking advantage of
different degrees of freedom.
The principle employed was that the creation of holes in the legs
of the AFM 10 would increase the flexibility of the tip 152,
thereby decreasing the tracking force, while retaining the
necessary strength for imaging. The process employed a commercially
available to create the holes 154, 156 in both arms of the tip 152.
FIGS. 13B-D shows the holes 154 created in the legs of the
cantilever tip 152. In the top panel, a single large hole 154 has
been created in each of the legs. A sequence of small holes 154
running the length of one of the legs is shown in the middle panel.
The lower panel shows the holes 154 of several sizes, and shows the
feasibility of accurately grouping a series if small holes 154
(note the sequence of eight small holes 154 created across the
width of the leg). Some detail is lost in the xerox as compared to
the original photographs.
Engineering principles can be used to design holes or spaces of
various shapes in the tip 152 that will best increase flexibility
and tracking force in whatever material is being used to construct
the tip 152. Laser technology is one convenient means to realize
these patterns in the material. The entire process can be automated
for commercial application and efficiency.
The ability to provide variable spring constant tips 14 can reduce
the hindering action of the fluid media during deflection of the
cantilever 12. This will enhance sensitivity and allow imaging in
high frequency. Other advantages include (1) the ability to cut
narrow wells into the cantilever tip 14 enabling the deposition of
a variety of chemical constituants, including pharmaceuticals,
conductive materials, bacteria and viruses; (2) the removal of
unnecessary extrusion parts of the cantilever 12 at the free end
near the tip attachment; such extrusion parts can interfere with
certain types of imaging; and (3) use in any kind of SFM of any
specimen including imaging with less loading force and less
specimen compression.
The following nonlimiting examples describe several exemplary
methods of preparing and utilizing the invention of the
Applicants.
EXAMPLE I
In the EPM 100, the specimen preferably moves while the cantilever
remains fixed. In FIG. 14A, the approach of the sample (in this
case negatively charged mica) towards the untreated Si.sub.3
N.sub.4 tip is indicated by arrow 1 at force=0. On contact with the
surface ("Contact"), the repulsive forces appear and increase
(arrow 2) as a function of decreasing distance. When the specimen
is withdrawn, the force decreases (arrow 3), and as the tip
disengages from the surface, an adhesive force (F.sub.adh)
reflecting the interaction between tip and specimen (in this case
about 2 nN) is noted. Note that this is beyond the force recorded
at the initial contact between tip and specimen. This image was
obtained while imaging in water. Dry imaging, not shown, has a
tenfold higher adhesion force. The capillary force which is a
prominent adhesion force in dry imaging disappears in fluid. A
polycationic lipid, lipfectamin (3:1 mixture w/w of
2,3-dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimetyhl-1-propanaminium
trifluoroacetate and dioleoyl phosphatidylethanolamine) and a
negatively charged lipid,
(1-palmitoyl-2-oleoyl-L-.alpha.-phosphatidyl-DL glycerol) were used
to alter the charge of the tip. In the following, the positively
charged tip is termed "DOSPA-tip" and the negatively charged
phosphatidylglycerol tip "pG tip." Lipids (0.5 ml, 10 mg/ml in
CHCL.sub.3) were dried in N.sub.2 in a glass test tube and
lyophilized overnight. Sodium phosphate buffer was used and the
incubate was sonicated until the solution became semitransparent. A
dry Si.sub.3 N.sub.4 tip was immersed into a preformed lipid
monolayer in the buffer-air interface. The surfaces scanned were
mica (negatively charged) and DEAE-Sephadex (positively charged)
beads.
A positively charged DOSPA-tip was used to scan negatively charged
mica in water. FIG. 14B shows the force curve as well as the bend
of the cantilever at forces equal to, greater and less than 0. Note
that at F=0, the cantilever is undeformed. As the distance between
tip and surface decreases (arrow 1), the cantilever tip is suddenly
pulled down by an attractive force with F<0 without much change
in Z due to the attraction between the positively charged tip and
the negatively charged surface. The maximum negative force at this
point is defined as the attractive force (F.sub.attr). As Z becomes
smaller, the cantilever bends reflecting an increase in repulsive
forces (arrow 2). On withdrawal, the tip force decreases (arrow 3)
and a very large adhesion force is noted (some 25 times that noted
for the untreated tip in FIG. 14A). Suddenly disengagement of the
tip occurs with a return to F=0 (arrows 4 and 5). A positively
charged DOSPA-tip was used to scan a positively charged surface
(DEAE-Sephadex on mica). FIG. 14C shows only the forces generated
by withdrawal of the sample from the cantilever and disengagement
(i.e., arrows 1 and 2 depicted in FIGS. 14A and B are not shown).
Note the large adhesion force when mica was the substrate prior to
disengagement (F.sub.adh) and the loss of most of the adhesion
force when DEAE-Sephadex coated the substrate as indicated by the
merging of arrow 3 into arrow 5.
Negatively charged pG tips were used to scan a negatively and
positively charged surface (mica and DEAE-Sephadex treated mica,
respectively). Opposite charges showed strong attractive and
adhesive forces, and like charges decreasing or eliminating these
forces. FIG. 14D shows again only the forces that occur on
withdrawing the sample surface from the tip. Note the large
adhesion force with the DEAE-Sephadex coated surface (F.sub.adh and
the return to F=0 as indicated by arrow 4) and the lack of adhesion
forces with mica (arrow 5).
The effect of NaCl on imaging mica was next analyzed. FIG. 14E
shows only the forces recorded on withdrawing the sample from the
tip. An untreated naked tip was used to image mica, and an adhesion
force of about 2 nN was in NaCl-free solution. 5M NaCl eliminated
the adhesion force (arrow 5 as compared to arrow 4). Our
nonlimiting interpretation of the data is that coulombic and van
der Waals forces are largely responsible for adhesion forces while
imaging mica with an unmodified tip. The coulombic and van der
Waals forces are dependent on the dielectric force of the solvent,
and the 5M NaCl increases the dielectric constant, thereby
decreasing the adhesion force.
The hydrophobic forces were next investigated. FIG. 14F shows only
the forces recorded on withdrawing the sample from the tip. Here,
nonpolar paraffin was the sample. Since paraffin is nonpolar, the
usually dominant coulombic forces are eliminated, and the van der
Waals forces are negligible as compared to the hydrophobic forces.
A negatively charged pG tip was used and the imaging was performed
in ethanol, water and 5M NaCl. No adhesion force was noted with
ethanol (arrow 5), but large and virtually identical adhesion
forces were noted with water and NaCl (F.sub.adh with a return to
F=0 as indicated by arrow 4). Our interpretation is that the
contact area is exposed to the medium as the tip is disengaging
from the specimen. Ethanol is randomly oriented on nonpolar
surfaces since it is also nonpolar, the decrease in entropy is
small, and the adhesion force will be minimal. Water has to be
ordered when in contact with a nonpolar, hydrophobic surface. With
the increase in the nonpolar surface on disengagement of the
cantilever, the entropy of water decreases since it must rearrange,
and the adhesion force is large. Water and 5M NaCl produce
virtually identical force curves on retraction, indicating that
charge does not play an important role which is in contrast to what
we observed imaging mica in 5M NaCl.
We then examined lysozymes adsorbed on a mica surface imaged in
phosphate buffer using a pG tip. (See FIGS. 9A and 9B.) The images
generated with the charged tips generally had a higher noise level
as compared to the images obtained with bare tips. A spectrum of
adhesion forces between 6 to 7 nN were recorded for the interaction
between the pG tip and the lysozymes which were much higher than
the 0.1 to 0.6 nN forces obtained for the interaction between naked
tips and lysozymes under the same condition. The small fluctuation
of the adhesion force probably reflected different residues of the
lysozyme molecule being probed. The variations in force may also be
due to the possibility of the probe touching a particular spot on
the lysozyme and the presence of multiple regions with similar
electrostatic states on the lysozyme.
Based on these results, (1) the cantilever is a very sensitive
detector of fields and forces at a molecular level and the
measurements were reproducible; (2) modification of the tip charge
is feasible permitting sensing of attractive, repulsive and
adhesive forces in the interactions between tips and surfaces of
differing charge and act according to theory; (3) the modified tips
are rugged; and (4) given the resolution obtained in our studies
with porin channels and the ACHR, we can image and measure forces
with sufficient resolution and sensitivity to attain the objectives
outlined.
EXAMPLE II
The nicotinic acetylcholine receptor (ACHR) was investigated since
it is one of the best characterized macromolecules involved in
synaptic transmission, can be readily imaged and the approach
should be generalizable to other receptors, channels and
interesting biomolecules. The ACHR from the electric organ of
Torpedo is probably the best characterized macromolecule involved
in synaptic communication, both functionally and biochemically. A
number of ligands are available, and the receptor closely resembles
the ACHR of the neuromuscular junction. It is thought to have a 255
kDa pentameric comply with an .alpha.2:.beta.;.gamma.:.delta.
stoichiometry, exists often as a dimer with a disulfide bridge
between the .delta. subunits of the adjacent monomers or as a
monomer. Each monomer has two nonequivalent acetylcholine binding
sites. The two .alpha. subunits are associated with
.alpha.-bungarotoxin (.alpha.-BTX) binding. The .alpha. subunits
are nonequivalent, are not adjacent as deduced from electron
microscopy (EM) and are separated by an angle of about 144.degree.
as deduced from immunoelectronmicroscopy. The current model for T.
California as observed from the synaptic side has been deduced to
be .alpha.:.beta.:.alpha.:.delta.:.gamma. (counterclockwise) from
immunoelectronmicroscopy and cross-linking experiments as well as
from analogy to EM studies of T. mannorata. When coexpressed in
fibroblasts, however, .alpha. and .beta. subunits do not associate
effectively and so do not bind ligand, whereas .alpha.-.delta. and
.alpha.-.gamma. do. The .gamma. subunit has a binding site for
wheat germ agglutinin (WGA). Several arrangements for the subunits
have been proposed, but the actual arrangement is uncertain. FIGS.
12A and B show the accepted arrangement of the subunits, the
location of the ACh binding site and the ion channel in the center,
and FIG. 12C is an AFM image of the AChR using an unmodified
cantilever tip. The pentameric structure of the ACHR receptor is
visible, and the intramolecular pore that enables the protein to
conduct cations is clearly identified. Of the five subunits, three
(marked as 1, 2, and 4) are bulkier than the other two. The
receptor has a diameter of about 113 .ANG. with a diameter at the
mouth of about 40 .ANG.. Of note is that the quaternary structure
of the ACHR shown here has higher resolution than the structural
data obtained by EM and x-ray crystallography. Acetylcholine is
attached to a neutral lipid which, in turn, is used to coat and
modify the cantilever tip. A large attractive and adhesive force
was observed with the EPM 100 allowing identification of the ACHR
and, subsequently, the subunit that contains the binding site. We
observed that: (1) the cantilever tip is a very sensitive detector
of molecular forces (e.g., repulsive, attractive and adhesion
forces); (2) modification of the tip charge is feasible permitting
sensing of attractive as well as repulsive and adhesion forces; (3)
the interactions between the tips of differing charge and surfaces
of differing charge are observable and act according to theory; (4)
the importance of hydrophobicity for a given environment can be
assessed by using a nonpolar surface; (5) the modified tips are
rugged; (6) the results are reproducible; and (7) we can assess the
morphology of a biomolecule (the ACHR) with sufficient resolution
to develop the biospecific probe.
EXAMPLE III
The biological gap junction was investigated by performing AFM on
gap junctions of a membrane (model shown of gap junction in FIG.
10). Increasing forces of 0.8 nN, 3.1 nN, 10.1 nN and a repeat scan
of 10.1 nN were made (see FIGS. 11A-E). The field size used was
1.5.times.1.5 microns and the line cut of each image is shown in
FIG. 11E. These line cuts show that the top membrane is removed at
higher forces, reducing the thickness of the structure to about
half the original value (15 nM to 7 nM in FIG. 11D). This shows the
"docking" portion of the gap junction and the six hexagonal array
is apparent from FIG. 11E.
While preferred embodiments of the invention have been shown and
described, it will be clear to those skilled in the art that
various changes and modifications can be made without departing
from the invention in the broader aspects as set forth in the
claims provided hereinafter.
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